Therapeutic and diagnostic methods and compositions for neurodegenerative diseases

ABSTRACT

Methods and compositions relating to motor neurons derived from induced pluripotent stem cells of subjects having a neurodegenerative disease, where the motor neurons exhibit phenotypes characteristic of the neurodegenerative disease, are provided herein. In particular, the present invention provides methods for screening putative therapeutic agents and methods for diagnosing living subjects as having a neurodegenerative disease. In addition, the present invention provides therapeutic gene transfer methods for treating or preventing a neurodegenerative disease in a subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/974,296, filed Apr. 2, 2014, which is incorporatedherein by reference as if set forth in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

Neurodegenerative disease is a term that encompasses a range ofpathologies that primarily affect neurons of the central nervous system.Neurodegenerative diseases are typically characterized by theprogressive degeneration and/or death of neurons in different regions ofthe nervous system and the resulting impairments in movement and mentalfunctioning. Neurodegenerative diseases, which are incurable and oftendebilitating, have an enormous impact on the lives of affectedindividuals and their families as well as society as a whole.

Parkinson's disease, amyotrophic lateral sclerosis (ALS), andAlzheimer's disease are the most well-known neurodegenerative diseases,but other conditions, such as Spinal Muscle Atrophy, Charcot-Marie-Toothdisease, Huntington's disease, spinocerebellar ataxias, Guillain-Barrésyndrome, Parkinson's disease-related disorders, and other motor neurondiseases belong to the same clinical group. Pathologies common togenetically inherited and sporadic cases of these neurodegenerativediseases are the accumulation of misfolded proteins, especiallyneurofilament (NF), and axonal degeneration. It remains unknown howprotein aggregation, mitochondrial dysfunction, glutamate toxicity, anddisrupted calcium homeostasis promote axonal degeneration or why theseprocesses selectively affect specific populations of neurons, such asmotor neurons in ALS, although some evidence suggests that proteinaggregates disrupt axonal transportation and consequently promoteretraction of motor neuron axonal degeneration before the loss of cellbodies.

Accordingly, there remains a need for a better understanding of theetiopathology of neurodegenerative diseases. In addition, there remainsa need in the art for methods for detecting neurodegenerative conditionsbefore clinical symptoms manifest, for facilitating accurate diagnosisof neurodegenerative disease, for monitoring disease progression, andfor identifying candidate therapeutic agents to slow, halt, or reverseneurodegeneration.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of diagnosingneurodegenerative disease in a subject. The method can comprise thesteps of obtaining induced pluripotent stem (iPS) cells from somaticcells of a subject, where the iPS cells are capable of differentiationinto neurons; culturing the iPS cells under conditions suitable todifferentiate the iPS cells into neurons; and detecting an indicator ofneurofilament aggregation or neurite degeneration in the iPScell-derived neurons, where increased neurofilament aggregation orneurite degeneration relative to neurons derived from iPS cells of anindividual not having neurodegenerative disease indicates that thesubject has a neurodegenerative disease, and thereby diagnosingneurodegenerative disease in the subject.

In some cases, the neurodegenerative disease can be selected from thegroup consisting of amyotrophic lateral sclerosis (ALS), Alzheimer'sDisease (AD), Parkinson's Disease (PD), a PD-related disorder,Huntington's Disease (HD), Charcot-Marie-Tooth disease (CMT),Spinocerebellar ataxia (SCA), Spinal Muscle Atrophy (SMA), andGuillain-Barré syndrome (GBS). The neurodegenerative disease can be ALS,SMA, or CMT, and the subject's iPS cells can differentiate into motorneurons. Detecting neurofilament aggregation can comprise determining alevel of NF-L mRNA in the subject's iPS cell-derived motor neurons. Insome cases, the method can further comprise detecting a level ofphosphorylated neurofilament in a biological sample of the subject. Thebiological sample of the subject can be cerebrospinal fluid. Theneurodegenerative disease can be Alzheimer's disease, and the subject'siPS cells can differentiate into glutamatergic neurons or cholinergicneurons. The neurodegenerative disease can be Parkinson's disease or aPD-related disorder, and the subject's iPS cells can differentiate intodopaminergic neurons. The neurodegenerative disease can bespinocerebellar ataxia and the subject's iPS cells can differentiateinto granular neurons.

In another aspect, the present invention provides a method of detectinga neurodegenerative disease in a subject. The method can comprise thesteps of determining a level of NF-L mRNA in neurons derived from iPScells obtained from somatic cells of a subject; relating the determinedlevel to a reference level of NF-L; and thereby detectingneurodegenerative disease in the subject based on a reduced level ofNF-L relative to the reference level. The neurodegenerative disease canbe selected from the group consisting of amyotrophic lateral sclerosis(ALS), Alzheimer's Disease (AD), Parkinson's Disease (PD), a PD-relateddisorder, Huntington's Disease (HD), Charcot-Marie-Tooth disease (CMT),Spinocerebellar ataxia (SCA), Spinal Muscle Atrophy (SMA), andGuillain-Barré syndrome (GBS). In some cases, the neurodegenerativedisease is ALS, where the neurons can be motor neurons, and where thereference level can be a level of NF-L mRNA in motor neurons derivedfrom iPS cells of an individual having ALS. The reduced level of NF-LmRNA can be at least 50% lower than the reference.

In a further aspect, the present invention provides a method fortreating a neurodegenerative disease in a subject in need thereof. Themethod can comprise administering one or more recombinant nucleic acidsequences encoding at least a portion of NF-L to the subject, where thenucleic acid sequences can be targeted to neurons, and where expressionof NF-L in targeted neurons can treat the neurodegenerative disease. Theneurodegenerative disease can be selected from the group consisting ofamyotrophic lateral sclerosis (ALS), Alzheimer's Disease (AD),Parkinson's Disease (PD), a PD-related disorder, Huntington's Disease(HD), Charcot-Marie-Tooth disease (CMT), Spinocerebellar ataxia (SCA),Spinal Muscle Atrophy (SMA), and Guillain-Barré syndrome (GBS). Thenucleic acid sequences can be administered in a vector. The vector canbe a virus or virus-derived. The virus can be selected from the groupconsisting of an adenovirus, retrovirus, herpes virus, andadeno-associated virus. The vector can be a replication defectiveadenovirus.

In another aspect, the present invention provides a method forprotecting against neurite degeneration in a subject in need thereof.The method can comprise administering one or more nucleic acid sequencesencoding NF-L to tissue of the subject, where the nucleic acid sequencesare targeted to neurons of the tissue, and where expression of NF-Lprotects the targeted neurons from neurite degeneration. The subject canhave been diagnosed or can be suspected of having a neurodegenerativedisease. The neurodegenerative disease can be selected from the groupconsisting of amyotrophic lateral sclerosis (ALS), Alzheimer's Disease(AD), Parkinson's Disease (PD), a PD-related disorder, Huntington'sDisease (HD), Charcot-Marie-Tooth disease (CMT), Spinocerebellar ataxia(SCA), Spinal Muscle Atrophy (SMA), and Guillain-Barré syndrome (GBS).The nucleic acid sequences can be administered in a vector. The vectoris a virus or virus-derived. The virus can be selected from the groupconsisting of an adenovirus, retrovirus, herpes virus, andadeno-associated virus.

In a further aspect, the present invention provides a method ofevaluating a candidate neuroprotective agent. The method can comprisethe steps of contacting a candidate neuroprotective agent to neuronsderived from induced pluripotent stem (iPS) cells obtained from somaticcells of a human subject having a neurodegenerative disease, where theneurons exhibit a phenotype typical of the neurodegenerative disease;and evaluating the contacted neurons for a neuroprotective effect of theagent relative to non-contacted iPS cell-derived neurons of the subject.The neurodegenerative disease can be selected from the group consistingof amyotrophic lateral sclerosis (ALS), Alzheimer's Disease (AD),Parkinson's Disease (PD), a PD-related disorder, Huntington's Disease(HD), Charcot-Marie-Tooth disease (CMT), Spinocerebellar ataxia (SCA),Spinal Muscle Atrophy (SMA), and Guillain-Barré syndrome (GBS). Theneuroprotective effect can be selected from the group consisting of areduction in severity of neurodegeneration, a delay in onset ofneurodegeneration, a reduction in severity of neurofilament (NF)aggregation, and increased motor neuron viability in vitro.

In another aspect, the present invention provides a recombinant nucleicacid molecule comprising a motor neuron-specific promoter operablylinked to a nucleic acid sequence encoding a human NF-L polypeptide. Theinvention also provides a vector comprising said nucleic acid molecule.The vector can be a plasmid. The vector can be a virus or virus-derived.The virus can be selected from the group consisting of an adenovirus,retrovirus, herpes virus, and adeno-associated virus. The vector can bea replication defective adenovirus.

In a further aspect, the present invention provides a kit for diagnosinga subject predisposed to or suspected of developing a neurodegenerativedisease or suffering from a neurodegenerative disease. The kit cancomprise at least one oligonucleotide primer capable of hybridizing to aat least a portion of a NF-L target nucleic acid; at least one referencecorresponding to a level of NF-L target nucleic acid; at least onebuffer or reagent; and a container. The at least one oligonucleotideprimer can comprise the nucleotide sequence of any of SEQ ID NO:7-8. Theneurodegenerative disease can be selected from the group consisting ofALS, SMA, and CMT.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1I demonstrate iPS cell generation, neural differentiation, andmutation correction. (FIG. 1A) Contrast image of iPS cell coloniesgenerated by Sendai virus. (FIG. 1B) Immunofluorescent image of NANOGexpression in D90A SOD1 iPS cells. (FIGS. 1C-1D) DNA sequencing showingheterozygous nucleotides (A/C) in D90A (C) and homozygous nucleotide Ain corrected (D90D) (D) SOD1 iPS cells. (FIG. 1E) Schematic protocol forMN and non-MN differentiation. 3c: 3 small molecular compounds(SB431542, LDN193189, and CHIR99021); Pur: purmorphamine; Cyclo:cyclopamine. (FIGS. 1F-1G) ALS (D90A) and genetically corrected ALS(D90D) iPS cells differentiated to OLIG2⁺ MN progenitors at day-14,MNX1⁺ postmitotic MNs at day-21, and CHAT⁺ maturing MNs at day-28.(FIGS. 1H-1I) Quantification of TUJ⁺ neuronal population among totalHoechst labeled (HO) cells (FIG. 1H) and MNX1⁺ MNs among neurons (FIG.1I). Scale bar=50 μm.

FIGS. 2A-2D demonstrate SOD1 expression and aggregation in iPSC-derivedneurons. (FIG. 2A) Allelic imbalance assay showing the ratio of mutant(A) and wt (C) copy of SOD1 transcripts in fibroblasts (FIB),reprogrammed stem cells (iPS), and their differentiated neuroepithelia(NEP), MN progenitors (MNP), MNs, and non-MNs. (FIG. 2B) RT-qPCRanalysis showing SOD1 mRNA expression in MNs and non-MNs. (FIG. 2C)Representative Western blots and relative SOD1 expression levels (toGAPDH) in MNs and non-MNs. (FIG. 2D) SOD1 immuno-EM in neurites,cytoplasm, nuclei, and mitochondria of MN and non-MN cultures.Arrows=clusters of gold particles. No contrast staining for ALS non-MNsto permit better views of fine gold particles. Scale bar=2 μm.

FIGS. 3A-3D depict neurofilament (NF) aggregates in ALS iPSC-derivedneurons. (FIG. 3A) Immunofluorescent images of NF-H, NF-M, and NF-L inCHAT⁺ MNs. NF staining in the insets is magnified on the right panel.Arrows indicate NF aggregates in the cell body; arrowheads indicate NFaggregates in neurites. Scale bar=50 μm. (FIG. 3B) EM showing NFarrangement in cell body (left) and neurites (right) of MN cultures.Scale bar=2 μm. (FIGS. 3C-3D) Quantification of NF aggregate-containingcell bodies (FIG. 3C) and neurites (FIG. 3D) in MNs and non-MNs atday-4, 7, and 10 after plating. *p<0.05.

FIGS. 4A-4G depict degenerative changes in ALS MN neurites. (FIG. 4A)Colorimetric measurement of LDH (normalized to gDNA) in culture mediafrom MN and non-MN cultures. (FIG. 4B) Cleaved caspase3 staining(arrows) and (FIG. 4C) quantification. (FIG. 4D) Phase contrast imagesof MNs and non-MNs at day-10. Arrows indicate bead-like swellings inneurites. Inset is magnified in upper-right. (FIG. 4E) Immunofluorescentimages of p-NF-H in MNs and non-MNs. Arrowheads indicate bead-likestructures in neurites. Inset magnified in upper right. (FIG. 4F)Quantification of beads on neurites. (FIG. 4G) ELISA quantification ofp-NF-H in media (normalized to gDNA). *p<0.01. Scale bar=50 μm.

FIGS. 5A-5G present images demonstrating NF aggregation and neuritedegeneration in neurons expressing D90A SOD1 in a wild-type (wt)background. (FIG. 5A) Western blots and relative expression of SOD1 (toGPDH) in MNs and non-MNs derived from hESCs expressing D90A SOD1 orEGFP. (FIG. 5B) Immunofluorescent images of NF-H, NF-M, and NF-L inCHAT⁺ cells from SOD1- and EGFP-expressing hESCs. NF staining in theinsets is magnified on the right panel. Arrows indicate NF aggregates inthe cell body; arrowheads indicate NF aggregates in neurites. (FIGS. 5C,5D) Quantification of NF aggregate-containing cell bodies (FIG. 5C) andneurites (FIG. 5D) at day-4, 7, and 10 after plating neurons. (FIG. 5E)Phase contrast images of MNs and non-MNs from mutant SOD1- andEGFP-expressing hESCs. Arrows indicate bead-like formations in neurites.Inset is magnified in upper-right. (FIGS. 5F, 5G) p-NF-H (FIG. 5F) andLDH (FIG. 5G) in culture media from MN and non-MN cultures derived fromSOD1- or EGFP-expressing hESCs. *p<0.05; **p<0.01. Scale bar=50 μm.

FIGS. 6A-6F present data demonstrating expression of NF subunits inneurons. (FIG. 6A) Relative levels of mRNAs for NF-L, NF-H, and NF-M,and β-actin mRNA in ALS (D90A) and corrected (D90D) MNs in the presenceof actinomycin D measured by RT-qPCR (p<0.05 between D90A and D90D).(FIG. 6B) Relative expression and representative Western blots of NF-H,NF-M, and NF-L in MNs and non-MNs as compared to neuron-specific enolase(NSE). (FIG. 6C) The proportion of NF-L among total NF protein in MNs(upper panel) and non-MNs (lower panel). *p<0.05 between ALS (D90A, A4V)and genetically corrected ALS (D90D) or wt (IMR-90-4) cells. (FIG. 6D)Relative levels of NF-L and β-actin mRNA in ALS (D90A) and corrected(D90D) MNs in the presence of actinomycin D measured by RT-qPCR (p<0.05between D90A and D90D). (FIG. 6E) Western blotting for mutant SOD1 in MNand non-MN samples pulled down by the 3′UTR NF-L mRNA probe. (FIG. 6F)Input samples were blotted for A5C3 antibody.

FIGS. 7A-7H present data demonstrating the effects of NF-L expression onNF aggregation and neurite degeneration in ALS MNs. (FIG. 7A) Westernblots and relative (to neuron-specific enolase (NSE)) expression ofNF-H, NF-M, and NF-L in MNs from GFP-, NF-L-expressing ALS (D90A) iPScells as well as genetically corrected (D90D1 & 2) ALS iPS cells. (FIG.7B) The proportion of NF-L among total NF protein in the presence of 1μg/ml of DOX. (C, D) Quantification of NF aggregate-containing cellbodies (FIG. 7C) and neurites (FIG. 7D) in MNs. (FIG. 7E) Phase contrastimages of MNs at day-10. Arrows indicate bead-like swellings inneurites. Inset is magnified in upper-right. (FIG. 7F) Immunofluorescentimages of pNF-H in MNs. Arrowheads indicate bead-like structures inneurites. (FIG. 7G) Quantification of bead-like formations. (FIG. 7H)ELISA quantification of pNF-H in media from MN cultures. Scale bar=50μm. *p<0.05.

FIGS. 8A-8C present data to demonstrate that sporadic ALS motor neuronsexhibit NF aggregates and changes in NF-L mRNA levels. (FIG. 8A)Immunofluorescent staining for NF-L in sporadic and control (IMR90) MNsat day-10. Inset is magnified on the right. Arrow indicates aggregate incell body and arrowhead indicates inclusion in neurite. (FIG. 8B)Electron micrograph shows NF aggregate in a MN neurite from ALS but notcontrol (IMR-90). (FIG. 8C) Quantification of NF-L mRNA by RT-qPCRbetween control (IMR-90) and sporadic ALS MNs.

FIGS. 9A-9B present (FIG. 9A) nucleotide (SEQ ID NO:1) and (FIG. 9B)amino acid (SEQ ID NO:2) sequences for NF-L.

FIGS. 10A-10B present (FIG. 10A) nucleotide (SEQ ID NO:3) and (FIG. 10B)amino acid (SEQ ID NO:4) sequence for NF-H.

FIGS. 11A-11B present (FIG. 11A) nucleotide (SEQ ID NO:5) and (FIG. 11B)amino acid (SEQ ID NO:6) sequences for NF-M.

FIGS. 12A-12F present immunofluorescent images of NF-H, NF-M, and NF-Lin CHAT motor neurons (MNs) and CHAT non-MN from wild-type (FIGS. 12A,12D), TDP43 mutant (FIGS. 12B, 12E), and sporadic ALS subjects (FIGS.12C, 12F). Staining in the insets is magnified on the right panel.Arrows indicate NF inclusions in the cell body; arrowheads indicate NFinclusions in neurites. Scale bar=50 μm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the Inventors'discovery that motor neurons differentiated from induced pluripotentstem cells of ALS patients exhibit phenotypes characteristic ofneurodegenerative diseases such as MN-selective accumulation ofneurofilament (NF) protein and axonal degeneration. The Inventorsfurther discovered that ALS-iPS cells exhibit altered proportions of NFsubunits and significantly reduced levels of NF-low (NF-L). Usinginduced pluripotent stem (iPS) cells derived from ALS patients,including those with genetic mutations (SOD1 and TDP43) and withoutgenetic mutations (sporadic), the Inventors discovered a motorneuron-selective NF aggregation at early stages and showed that thesemotor neurons gradually undergo axonal degeneration. They furtherdiscovered that ALS motor neurons show reduced expression ofneurofilament-low (NF-L) and altered proportion of NF subunits (low,medium and high molecular weight). Importantly, they found thatcorrection of NF-L mRNA expression in motor neurons restores the regularproportion of NF subunits, prevents NF aggregation, and subsequentlyprotects motor neurons from undergoing degeneration.

To date, animal models have failed to fully recapitulate theneuropathology observed in human patients having neurodegenerativediseases. As described herein, ALS-iPS cells retain the ALS diseasephenotype and have the capacity for differentiation into neuronal cellsthat exhibit a phenotype typical of ALS. For example, motor neuronsderived from ALS-iPS cells undergo continual degeneration over time. TheExamples below disclose that iPS cells generated from a human patientwith ALS can be differentiated into motor neurons that then exhibitdisease-specific phenotypes and undergo disease-specific cell death inthe culture dish. Accordingly, the work described herein represents avery powerful example of a model in which to explore neurodegenerativedisease mechanisms and to screen for novel compounds that may attenuateor block neurodegenerative disease processes.

In the specification and in the claims, the terms “including” and“comprising” are open-ended terms and should be interpreted to mean“including, but not limited to . . . . ” These terms encompass the morerestrictive terms “consisting essentially of” and “consisting of.”

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. As well, the terms “a” (or “an”), “one or more” and “at leastone” can be used interchangeably herein. It is also to be noted that theterms “comprising”, “including”, “characterized by” and “having” can beused interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. All publications and patentsspecifically mentioned herein are incorporated by reference in theirentirety for all purposes including describing and disclosing thechemicals, instruments, statistical analyses and methodologies which arereported in the publications which might be used in connection with theinvention. All references cited in this specification are to be taken asindicative of the level of skill in the art.

Methods of the Invention a. Treatment Methods

One aspect of the present invention relates to methods for treating orpreventing a neurodegenerative disease and for protecting against axonaldegeneration in a subject in need thereof. As used herein, the terms“treat” and “treating” refer to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) an undesired physiological change or disorder,such as progressive neurodegeneration. For purposes of this invention,beneficial or desired clinical results include, without limitation, analleviation of one or more clinical indications, decreased motor neurondegeneration, reduced severity of one or more clinical indications,diminishment of the extent of disease, stabilization of the diseasestate (i.e., not worsening), delay or slowing, halting, or reversingneurodegenerative disease progression, and partial or completeremission, whether detectable or undetectable. “Treatment” also refersto prolonging survival by weeks, months, or years as compared toexpected survival if not receiving treatment according to a methodprovided herein. Subjects in need of treatment can include those alreadyhaving or diagnosed with a neurodegenerative condition or disorder aswell as those prone to, likely to develop, or suspected of having theneurodegenerative condition.

As used herein, the term “neurodegenerative disease” refers to a diseaseor disorder affecting nerves of the central nervous system thattypically manifests as one or a combination of motor, sensory,sensorimotor, or autonomic neural dysfunction. A neurodegenerativedisease can be sporadic or genetically acquired, can result fromsystemic disease or traumatic injury, or can be induced by a neurotoxicagent such as a chemotherapeutic agent. In exemplary embodiments, aneurodegenerative disease appropriate for the present invention is oneassociated with or characterized, at least in part, by neurofilament(NF) aggregation and neurite degeneration. Neurodegenerative diseasesappropriate for the present invention include, without limitation,Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease(HD), Spinal Muscle Atrophy (SMA), spinocerebellar ataxias (SCA),Guillain-Barré syndrome (GBS), Parkinson's disease-related disorders,Charcot-Marie-Tooth disease (CMT), amyotrophic lateral sclerosis (ALS),and other motor neuron diseases.

Huntington's Disease (HD) is characterized by progressive neuronal deathin different areas of the brain and the appearance of motor coordinationdeficits and hyperkinetic movement disorders. The spinocerebellarataxias (SCA) are a group of neurodegenerative diseases characterized bycerebellar ataxia (deficit or failure of muscular coordination),occulomotor abnormalities, upper and lower motor neuron signs, cognitivedecline, epilepsy, autonomic dysfunction, sensory deficits, andpsychiatric manifestations. See, e.g., Schols et al., Lancet Neurol3(5):291-304 (2004). Charcot-Marie-Tooth disease (CMT) (also known asHereditary Motor and Sensory Neuropathy, “HMSN”) is the most commoninherited disorder of the peripheral nervous system, affectingapproximately 1 in 2500 individuals. Clinical subtypes of CMT areassociated with progressive distal muscle weakness, atrophy,demyelinating neuropathy (CMT type 1), and axonal loss (CMT type 2).

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron (MN)disease having no effective treatment (Robberecht and Philips, Nat. Rev.Neurosci. 14:248-264 (2013)). A clinical diagnosis of ALS, defined byprogressive signs and symptoms of upper and lower motor neurondysfunction, is typically confirmed using electromyography. Additionaltests such as magnetic resonance imaging (MRI) of the brain or spinalcolumn can exclude other diseases. In the absence of treatments toeffectively delay or halt disease progression, ALS patient care islargely palliative. While mostly sporadic, some cases of ALS areassociated with genetic mutations, among which 20% is caused bymutations in the copper zinc superoxide dismutase (SOD1) gene.

In some cases, the present invention provides a gene therapy method fortreating a neurodegenerative disease in a subject in need thereof, wherethe neurodegenerative disease affects motor neurons or is associatedwith motor neuron degeneration. The primary goal of gene therapy is totreat a loss-of-function genetic disorder by delivering correctingtherapeutic DNA sequences into the nucleus of a cell, whereby long-termexpression of such therapeutic DNA sequences at physiologically relevantlevels can partially or fully correct the loss-of-function phenotype.Therapeutic gene transfer offers potential advantages over directadministration of a polypeptide systemically or via continuous ortargeted production of a target gene product in vivo. Embodiments of theinvention comprise delivery of a gene therapy vector having aheterologous gene of interest to obtain stable gene expression in targetcells or tissues. The method can include contacting a cell or tissue ofa subject in need thereof to a vector comprising a heterologous gene,wherein the vector is introduced into a motor neuron of the subject andwherein expression of the heterologous gene treats the neurodegenerativedisease. In some cases, the subject has been diagnosed or is suspectedof having amyotrophic lateral sclerosis (ALS) or spinal muscular atrophyof infancy.

As used herein, the phrase “expression of the heterologous gene” refersto expression of a therapeutically effective amount of a heterologousgene in a cell or cells to which the gene therapy vector has beenintroduced. The “heterologous” gene may be a second copy or an alteredcopy of a gene that is already part of the subject's genome. Continuousin situ production of physiological concentration of the gene productcan provide a therapeutically effective amount of such molecules,thereby treating the neurodegenerative disease. In some cases, theheterologous gene encodes NF-68(L) protein (“NF-L”). Nucleotide andamino acid sequences for NF-L are set forth as SEQ ID NO:1 and SEQ IDNO:2, respectively. In such cases, introducing a viral gene therapyvector comprising nucleic acid sequence encoding NF-L polypeptide, andtargeting the vector to one or more types of neurons, can increaseexpression of NF-L mRNA and the proportion of NF-L subunit to relativeto subunits NF-200(H) (“NF-H”) and NF-145(M) (“NF-M”), whereby theneurodegenerative disease is treated. Nucleotide and amino acidsequences for NF-H are set forth as SEQ ID NO:3 and SEQ ID NO:4,respectively. Nucleotide and amino acid sequences for NF-M are set forthas SEQ ID NO:5 and SEQ ID NO:6, respectively.

Targeted delivery of a therapeutically effective amount of aheterologous gene can treat, prevent, reverse, remove, or compensate forNF aggregation or neurite degeneration in susceptible motor neurons.Accordingly, targeted delivery of a therapeutic gene such as NF-L tovulnerable neurons of the central nervous system or peripheral nervoussystem can be a useful neuroprotective (e.g., prophylactic) ortherapeutic strategy for a subject having, suspected of having,predisposed to developing, or likely to be susceptible to aneurodegenerative disease.

In exemplary embodiments, recombinant nucleic acid sequences areadministered in a vector. Vectors appropriate for use according to amethod of the present invention include, without limitation, viralvectors, preferably adenoviruses, herpes viruses, adeno-associatedviruses (AAV), retroviruses including lentiviruses, poxviridae,baculovirus, vaccinia or Epstein-Barr viruses. In exemplary embodiments,vectors are adenoviruses. Various serotypes of adenovirus are known inthe art. For example, an appropriate adenovirus serotype for useaccording to a method of the present invention can be a type 2 or type 5human adenovirus (Ad2 or Ad5) or an adenovirus of animal origin (e.g.,adenoviruses of bovine, canine, murine, ovine, porcine, avian, andsimian origin). In some cases, it is preferable to usereplication-defective adenoviruses, comprising at least onenon-functional viral region selected from E1, E2 and/or E4. Suchadenoviruses may be produced according to conventional methods such asthe method described by Dedieu et al., J. Virology 71:4626-4637 (1997).Recombinant adenoviruses carrying a replication defective genome can beprepared according to methods known in the art using either competentpackaging cells or transient transfection (Graham F. L. and Prevec L.,Gene transfer and expression protocols; Manipulation of adenovirusvectors, Methods in Molecular Biology (1991), The Humana Press Inc,Cliften, N.J., chapter 11, pp. 109-128). In some cases, a method oftreating a neurodegenerative disease in a subject in need thereofcomprises contacting a cell or tissue of a subject to a recombinantadeno-associated virus vector comprising a heterologous gene, whereinthe vector is introduced into a neuron (e.g., motor neuron) of thesubject and wherein expression of the heterologous gene treats theneurodegenerative disease.

In another aspect, the present invention provides a method of protectingagainst axonal or neurite degeneration in a subject in need thereof. Themethod comprises administering one or more recombinant nucleic acidsequences (polynucleotides) encoding a NF-L polypeptide to tissue of thesubject, wherein the nucleic acid sequences are targeted to, forexample, neurons of the tissue (e.g., motor neurons), and whereinexpression of NF-L in targeted neurons protects the motor neurons fromneurite degeneration. In some cases, the subject has been diagnosed oris suspected of having ALS or spinal muscular atrophy of infancy.Recombinant nucleic acid sequences can be administered in a vector suchas a viral vector or virus-derived vector. Viral vectors appropriate foruse according to a method provided herein include, without limitation,adenoviruses, retroviruses, herpes viruses, and adeno-associatedviruses. In some cases, a motor neuron-specific promoter can be used todrive expression of a therapeutic target gene such as NF-L. The efficacyof gene therapy may be monitored by clinical assessment as well asmeasurement of phosphorylated neurofilament levels as reduction ofaxonal (neurite) degeneration will decrease the release ofphosphorylated neurofilament.

b. Diagnostic Methods

In another aspect, the present invention provides a method of diagnosinga subject as having a neurodegenerative disease. The method can comprisedetecting neurofilament protein aggregation, neurite degeneration,and/or cell death as an indicator of neurodegenerative disease in thesubject. For example, a method of the present invention can comprise (a)obtaining induced pluripotent stem (iPS) cells from somatic cells of asubject, wherein the iPS cells are capable of differentiation into motorneurons; (b) culturing the iPS cells under conditions suitable todifferentiate the iPS cells into motor neurons; and (c) detectingneurofilament levels and aggregation or neurite degeneration in the iPScell-derived motor neurons, where reduced neurofilament levels, orneurofilament aggregation or neurite degeneration indicates that thesubject has a neurodegenerative disease.

In exemplary embodiments, the subject is a living human. For example,the subject can be a living human suspected of or at risk for developinga neurodegenerative disorder. Skin biopsy or blood samples can beobtained from such individuals and reprogrammed into motor neuronseither directly (Vierbuchen et al., Nature 463:1035-1041; Son et al.,Cell Stem Cell 9:205-218) or indirectly via iPS cells as exemplifiedherein. An individual's (e.g., human patient) motor neurons can beassayed for reduced levels of NF-L polypeptide or mRNA, increasedrelease of phosphorylated neurofilament to culture media, orneurofilament aggregation, neurite degeneration, susceptibility tostress, or increased cell death. NF-L levels can be assayed using, forexample, RT-PCR (for mRNA) or using an anti-NF-L antibody (e.g., Westernblot). Release of soluble phosphorylated NF into culture medium can bedetected using, for example, an enzyme-linked immunosorbent assay(ELISA). Such assays could be readily performed in a clinical setting.In some cases, diagnosis of an individual suspected of or at risk fordeveloping a neurodegenerative disorder may be validated by detectingincreased levels of phosphorylated neurofilament in a cerebral spinalfluid sample obtained from the individual.

c. Screening Methods

In another aspect, the present invention provides methods foridentifying candidate therapeutic agents to treat a neurodegenerativedisease, to slow or halt neurodegeneration, to alter a neurodegenerativedisease mechanism, or to correct an observed neurodegenerative diseasephenotype. For example, methods of the present invention can comprisetesting compounds for their ability to modify or restore cellular levelsof NF-L, to restore cellular proportions of NF subunits, or to attenuateor prevent neurite degeneration. Alternatively, the methods providedherein could identify compounds that promote cell survival (e.g.,compounds that stimulate intracellular protective pathways or promotesecretion of growth factors) independent of NF function.

In some cases, the present invention provides a method of evaluating acandidate neuroprotective agent, where the method comprises the steps ofcontacting a candidate neuroprotective agent to motor neurons derivedfrom induced pluripotent stem (iPS) cells obtained from somatic cells ofa human amyotrophic lateral sclerosis (ALS) patient, wherein the motorneurons exhibit a phenotype typical of ALS; and evaluating the contactedmotor neurons for a neuroprotective effect of the agent. In someembodiments, the method will include evaluating the effect of the agentrelative to motor neurons derived from iPS cells obtained from somaticcells of a human ALS patient that have not contacted the agent. A“neuroprotective effect” can include, without limitation, a reduction inseverity of neurodegeneration, a delay in onset of neurodegeneration, areduction in severity of neurofilament (NF) aggregation and neuritedegeneration, and increased motor neuron viability in vitro. Forexample, a compound contacted to an ALS iPS-derived motor neuron culturecan alter neurofilament levels, neurofilament subunit proportion,neurofilament aggregation, axonal or neurite degeneration, or cellsurvival and such effects of the compound can be assayed, selected, andvalidated. See, for example, Yang et al., Cell Stem Cell. (2013) Boyd etal., J Biomol. Screen. 19(1):44-56 (2014); Naohiro et al., Sci. Transl.Med. 145:104 (2012); Sharma et al., Methods Enzymol. 506:331-60 (2012);Burkhardt et al., Mol. Cell Neurosci. 56:355-64 (2013).

In some cases, the method can comprise differentiating iPS cells,derived from somatic cells of a human subject, into neurons, preferablyas disclosed below, and examining the effect of a test compound on NFsubunit proportions or NF-L levels of the motor neurons, where anincrease in NF-L protein relative to motor neurons derived from iPScells from somatic cells of an ALS patient that have not been exposed tothe test compound indicates that the compound modifies cellular NFsubunit proportions or NF-L levels. As used herein, the phrases“differentiation into neurons” and “differentiating into neurons” referto promoting the differentiation of iPS cells into cells having thegenetic markers, cell function, and cell morphology characteristic of aparticular neuronal lineage (e.g., motor neurons, Gabaergic neurons,cholinergic neurons, dopaminergic neurons).

The method can additionally or alternatively comprise examining theeffect of a test compound on axonal or neurite degeneration relative toneurons derived from iPS cells from somatic cells of a human subjecthaving a neurodegenerative disease that have not been exposed to thetest compound. In some cases, the method comprises examining the effectof a test compound on axonal or neurite degeneration relative to motorneurons derived from iPS cells of a human ALS patient that have not beenexposed to the test compound. Such effects on ALS-patient derived motorneurons can be detected by assaying for NF-L protein production, NF-LmRNA levels, degree of neurite degeneration, and onset or extent of celldeath. Changes in neurite length are detected as an indicator of neuritedegeneration. In exemplary embodiments, neurite length is measured usinga reporter system such as the luciferase reporter NanoLuc (Nluc) fusedwith SYNAPTOPHYSIN (SYP), a synaptic glycoprotein that targets the Nlucreporter to axonal membrane, as described in U.S. Patent ApplicationSer. No. 62/112,441, filed Feb. 5, 2015 (incorporated herein byreference in its entirety).

In another embodiment, the method includes screening test compounds foran effect on motor neuron survival. Compounds that increase motor neuroncell survival, relative to ALS-patient derived motor neurons that havenot been exposed to the test compound, are excellent candidates forfurther drug testing.

In some cases, the method further comprises obtaining a population ofiPS cells derived from somatic cells from human subject known to have aneurodegenerative disease such as ALS. In exemplary embodiments, thesomatic cells of an ALS patient are fibroblasts. Other types of somaticcells may also be used, including without limitation, blood cells, hairfollicle cells, fat cells, and neural cells. To confirm thatreprogramming of wild-type and ALS fibroblasts to a pluripotent statehas occurred, one may wish to use standard techniques including, but notlimited to, quantitative PCR with reverse transcription (qRT-PCR),teratoma formation, DNA fingerprinting and microarray analysis.Pluripotency criteria have been described (see Chan et al., Live cellimaging distinguishes bona fide human iPS cells from partiallyreprogrammed cells, Nat. Biotech., 27:1033-1037, 2009). Typically, onewould look for teratoma formation and expression of endogenous Oct4,SSEA, TRA, and other markers. One could also assay for the ability ofthe cells to make embryoid bodies that produce all three dermallineages.

Compositions of the Invention

In a further aspect, the present invention provides compositions usefulfor treating a neurodegenerative disease or protecting against neuritedegeneration in a subject in need thereof. In particular, the presentinvention provides recombinant nucleic acid molecules and expressionvectors comprising such nucleic acid molecules. In some cases, arecombinant nucleic acid molecule comprises a nucleic acid sequenceencoding a NF-L polypeptide (e.g., human NF-L). A recombinant nucleicacid molecule of the invention can further comprise a motorneuron-specific promoter operably linked to a target nucleic acidsequence (e.g., a nucleic acid sequence encoding human NF-L).

In exemplary embodiments, a recombinant nucleic acid molecule of thepresent invention is in a vector. In some cases, the vector is a plasmid(e.g., plasmid expression vector) or is a virus or virus-derived. Forexample, the vector can be a virus selected from the following: anadenovirus, a retrovirus, a herpes virus, and an adeno-associated virus.In some cases, the vector is a replication-defective adenovirus vector(e.g., a human replication-defective adenovirus). Areplication-defective adenovirus vector is capable of delivering itsgenome to an infected cell, but comprises one or more disablingmutations that prevent activation of viral early gene expression and DNAreplication. For example, the replication-defective adenovirus vectorpAdRSVβgal lacks the early region 1 (E1) genes needed to efficientlyactivate transcription of the other viral early genes, including thoseencoding viral DNA replication proteins and those responsible forinactivating the cellular DNA damage response. See Stratford-Perncaudetet al., J. Clin. Invest. 90:626-30 (1992).

In another aspect, the present invention provides kits useful for thediagnosis, treatment, or monitoring of a neurodegenerative disease. Forexample, a kit of the present invention can be used to diagnose ormonitor disease progression in a subject predisposed to or suspected ofdeveloping a neurodegenerative disease or suffering from aneurodegenerative disease. In some cases, a kit of the present inventioncan comprise the following components: at least one oligonucleotideprimer capable of hybridizing to or amplifying a target NF-L nucleicacid sequence; at least one reference corresponding to a level of NF-Ltarget nucleic acid; at least one buffer or reagent; and a container. Byway of example, oligonucleotide primers appropriate for a kit of thepresent invention include, without limitation, the nucleotide sequencesof SEQ ID NO:7 (5′-ATGAGTTCCTTCAGCTACGAGC-3′) and SEQ ID NO:8(5′-CTGGGCATCAACGATCCAGA-3′).

While the present invention is susceptible to various modifications andalternative forms, specific embodiments thereof have been shown by wayof example in the drawings and are herein described in detail. It shouldbe understood, however, that the description herein of specificembodiments is not intended to limit the invention to the particularforms disclosed, but on the contrary, the intention is to cover allmodifications, equivalents and alternatives falling within the spiritand scope of the invention as defined by the appended claims.

The invention will be more fully understood upon consideration of thefollowing non-limiting Examples.

EXAMPLES Example 1 Efficient Differentiation of ALS iPS Cells andGenetically Corrected ALS iPS Cells to Motor Neurons

Fibroblasts from a 50-year old female carrying the D90A SOD1 mutation(ND29149, P3; Coriell Institute) were reprogrammed using thenon-integrating Sendai virus as described (Ban et al., Proc. Natl. Acad.Sci. U.S.A 108:14234-14239 (2011)). A4V SOD1 mutant iPSC lines,established with retrovirus, were obtained from Coriell (ND35671). TheIMR-90-4 iPSC line, generated from fetal fibroblasts (Hu et al., 2010),was used as a wild-type (wt) control. The iPS cells reprogrammed withSendai virus were integration-free as confirmed by qPCR analysis forviral sequences (data not shown). All iPS cells became stable celllines, exhibited typical morphology, expressed the pluripotency markersincluding NANOG (FIGS. 1A-1B), OCT4, SSEA-4, and SOX2, generatedteratomas in vivo, and retained karyotype stability (some data notshown). Human ESCs (H9 line, NIH registry 0046) were used as anadditional wt control and as a recipient for transgenic expression ofmutant SOD1. The SOD1 D90A mutation was maintained as confirmed bysequencing exon 4 of SOD1 (FIG. 1C, 1D).

To establish isogenic controls under the same genetic and epigeneticbackground, we corrected the D90A mutation in the iPS cells using TALENmediated homologous recombination. Transgenes were targeted to the AAVS1locus in hESCs and iPSCs by TALEN. The donor plasmid for constitutiveexpression of D90A SOD1 was constructed by replacing the CAG-GFPcassette of the plasmid AAV-CAGGS-EGFP (Addgene, Cambridge, Mass.) withthe CAG-D90A SOD1 cassette via SpeI and MluI double digestion.Similarly, the Tet-inducible expression plasmid was constructed byreplacing the CAG-GFP cassette with the CAG-Tet-On 3G cassette(pTRE3G-SV40-polyA cassette, Clontech, Mountain View, Calif.) via theSpeI site. NF-L cDNA sequence was inserted into the SalI and MluI sitesof the plasmid for conditional NF-L expression. To correct the SOD1 D90Amutation, a 986 basepair (bp) or 829 bp fragment at both sides of theTALEN target point was PCR-amplified using genomic DNA from H9 hESCs.The digested fragments were then cloned into the multiple clone site ofplasmid PL452 (Frederick National Lab). TALENs pairs targeting the AAVS1locus was designed as described (Hockemeyer et al., Nat. Biotechnol.29:731-734 (2011)). TALEN activity was assayed via surveyor nuclease(Transgenomic, Omaha, Nebr.). A standard protocol was used for cellulartransfection and cloning as described (Hockemeyer et al., Nat.Biotechnol. 29:731-734 (2011)).

PCR analysis of individual single-cell-derived G418-resistant clonesusing a primer external to the 5′ donor homology region and a primeragainst the PGK promoter demonstrated disruption of the genomic locusand integration of the donor vector with a frequency of about 45% (datanot shown). Two selected clones, designated as D90D1 and D90D2, showedsuccessful correction of the targeted locus, as revealed by TaqMan SNPgenotyping assays and by sequencing (FIG. 1C, 1D). The corrected iPSCsdisplayed a normal karyotype, uniform expression of pluripotencymarkers, and generation of teratomas, and identical polymorphisms totheir parental cells as assayed by short tandem repeat analysis.

Pluripotent stem cells were first differentiated to neuroepithelia in aneural medium consisting of DMEM/F12, N2 supplement, and non-essentialamino acids in the presence of SB431542 (2 μM), LDN193189 (300 nM), andCHIR99021 (3 μM; Stemgent) for 7 days. At day 8, the neuroepithelia weretreated with RA (0.1 μM) and purmorphamine (0.5 μM) for 7 days for MNinduction. For generation of non-MNs, cyclopamine (0.5 μM) was added inplace of purmorphamine. At day-14, both MN and non-MN progenitors in theform of rosettes were isolated and expanded as floating clusters insuspension in the same respective medium but without SB431542,LDN193189, and CHIR99021 for an additional 7 days before plating onlaminin substrate for generating mature neurons. To generatesynchronized postmitotic neurons, the cultures were treated from day18-21 with compound E (0.1 μM) to block cell proliferation.

Initial experiments were designed to assay whether MN generation isaltered by SOD1 mutations. Using a protocol (FIG. 1E) modified fromprevious methods (Amoroso et al., J. Neurosci. 33:574-586 (2013)); Li etal., Stem Cells 26:886-893 (2008)), it was determined that both themutant SOD1 iPSCs (D90A and A4V) and genetically corrected (D90D), aswell as wt iPSCs (IMR-90-4), efficiently differentiated into OLIG2⁺ MNprogenitors at day 14, MNX1⁺ postmitotic MNs at day 21, and CHAT⁺maturing MNs by 21-30 days (FIGS. 1F-1G), with 94% of the TUJ1⁺ neurons,or 90% of total differentiated cells being MNX1⁺ MNs (FIG. 1H-I). Theseresults indicated that ALS mutations do not affect MN development. Wealso differentiated the iPSCs to spinal neurons that were void of MNX1⁺or CHAT⁺ MNs by blocking hedgehog signaling using cyclopamine duringneural patterning (FIG. 1E), which forms a non-MN control within theindividual iPSC line. At 4-5 weeks of iPSC differentiation, no glialfibrillary acidic protein (GFAP)-expressing astrocytes were observed(FIG. 1F-G), which is a similar result to previous observations thatGFAP-expressing astrocytes do not usually appear until after 2-3 monthsof hPSC differentiation (Krencik et al., Nat. Biotechnol. 29:528-534(2011); Serio et al., Proc. Natl. Acad. Sci. U.S.A 110:4697-4702(2013)). Additionally, we used compound E, a notch signaling inhibitor,to prevent generation of new neurons from progenitors. The highlyenriched and synchronized MNs and non-MNs enable phenotypiccharacterization at cellular and molecular levels.

Example 2 ALS Motor Neurons Exhibit Small Aggregates of Mutant SOD1 andNF Aggregates

Expression of multiple copies of disease-causing mutant SOD1 in animalsresults in increased expression and aggregation of SOD1 (Bruijn et al.,Science 281:1851-1854 (1998); Furukawa et al., Proc. Natl. Acad. Sci.U.S.A. 103:7148-7153 (2006); Karch et al., Proc. Natl. Acad. Sci. U.S.A.106:7774-9 (2009)). We first asked if SOD1 is comparably expressed inMNs and non-MNs. Western blotting indicated that the SOD1 level inday-30 MNs was approximately 40% lower than that in non-MNs (FIG. 2C).Next, we assayed whether a single copy of mutant SOD1 would alter thelevel of SOD1 and cause its aggregation in human ALS neurons (neurons ofhuman subjects having ALS). By quantitative PCR (qPCR), it was observedthat the ratios between the mutant and wild-type (wt) alleles were about1 in fibroblasts, iPSCs, neuroepithelia, MN progenitors, MNs, andnon-MNs for the D90A mutant, whereas in wt cells both copies were wt(FIG. 2A), indicating that the mutant SOD1 copy is maintained duringreprogramming and neural differentiation. By RT-qPCR we then found thatmutant SOD1 MNs and non-MNs expressed a similar SOD1 level as wt andgenetically corrected ALS cells (FIG. 2B). Interestingly, Westernblotting indicated that mutant MNs, but not mutant non-MNs, expressed aneven lower level of SOD1 than wt and genetically corrected ALS MNs (FIG.2C). This unexpected pattern of changes was replicated in six sets ofbiological samples. Thus, unlike in transgenic animals, the total amountSOD1 protein does not increase in ALS patients' MNs, at least at the ageanalyzed.

Immunostaining for SOD1 and examination under confocal microscopy showeda ubiquitous expression pattern without discernible aggregates in MNs.We reasoned that aggregates may have been absent or too small to discernunder light microscopy. Immuno-electron microscopy (EM) revealed an evendistribution of fine gold particles in cytoplasm and neurites of MNcultures from wt and genetically corrected ALS cells. In the ALS MNcultures, clusters of gold particles, averaged 64±5 nm in diameter, werepresent in cytoplasm, neurites, and nuclei (FIG. 2D). In transgenicanimals, SOD1 aggregates are often present on mitochondrial membrane(Bergemalm et al., J. Neurosci. 26:4147-4154 (2006); Pasinelli et al.,Neuron 43:19-30 (2004); Vijayvergiya et al., J. Neurosci. 25:2463-2470(2005)). Careful examination revealed no association of SOD1 aggregateswith mitochondria in ALS patients' MNs (FIG. 2D). In the ALS non-MNcultures, there were more gold particles than in MNs, but they weresingular, of 10.73±1.07 nm in diameter (FIG. 2D, the fine particles wererevealed in non-contrasted image for non-MNs). Thus, small SOD1aggregates are present in the cytoplasm, nuclei, and neurites but not inmitochondria of ALS MNs.

NF aggregates in the perikaryon and proximal axons of spinal MNs ishallmark ALS pathology (Carpenter, Neurology 18:841-851 (1968); Hiranoet al., J. Neuropathol. Exp. Neurol. 43:471-480 (1984)). Immunostainingfor NF-200(H), NF-145(M) and NF-68(L) at day-30 revealed distinct, focalaccumulation of immunoreactive products in cytoplasm and neurites ofCHAT⁺ ALS MNs (FIG. 3A). Such NF aggregates were rare in ALS non-MNs orMNs from wt and genetically corrected ALS iPSCs (see FIG. 3A). Theaggregates in the cytoplasm and proximal neurites were often accompaniedby lower immunofluorescent staining for NF (FIG. 3A) and other proteins(CHAT) in distal neurites. However, the distribution of βIII-tubulin inALS MN neurites was not altered. The identity of the aggregates wasconfirmed by electron microscopy, showing disorganized NFs near thenucleus or in the proximal neurite with mitochondria surrounding orinside of the aggregate (FIG. 3B).

NF aggregates were defined as a focal accumulation of immunoreactiveproducts with its intensity being 3 times higher than that in itssurroundings. Neurite swelling or beading was defined as an enlargementof a neurite that is at least twice the diameter of the neurite. Atleast 500 neurites were counted in each group. Statistical analyses wereperformed using one-way ANOVA (Tukey or Dunnett for multiplecomparisons) in SPSS13.0. Quantification of NF aggregates indicated anincreasing number of ALS MNs and their neurites that contained NFaggregates at day-24, 27, and 30, or 4, 7, and 10 days after platingday-21 cells for maturation (FIGS. 3C-3D). By day-10, over 60% of the MNcell bodies or 25% of neurites contained NF aggregates and the averagesize of the NF aggregate was 42.4-75.4 μm² in cell bodies and 1.75-5.53μm² in neurites. The wt and genetically corrected ALS MNs also containedan increasing number of NF aggregates over culture but at asignificantly less degree, reaching about 20% of the cells and 8% of theneurites. Fewer NF aggregates existed in non-MNs as compared to MNs inthe ALS cells. Thus, NF aggregates are preferentially present in ALS MNsand over time more MNs contain aggregates.

In or surrounding the NF aggregates were often accumulation ofmitochondria (FIG. 3B). In SOD1 transgenic mice, mitochondria are oftenswollen or contain vacuous formations (Vijayvergiya et al., J. Neurosci.25:2463-2470 (2005); Gurney et al., Science 264:1772-1775 (1994); Wonget al., Neuron 14:1105-1116 (1995); Liu et al., Neuron 43:5-17 (2004);Bergemalm et al., J. Neurosci. 26:4147-4154 (2006)). This, however, wasnot observed in the ALS human MNs (FIG. 3B) despite the presence of NFaggregates.

Example 3 Neurite Degeneration in ALS Motor Neurons

Since ALS MNs contain SOD1 aggregates and NF aggregates, we asked ifthese neurons undergo degeneration or cell death. Measurement of lactatedehydrogenase (LDH), a soluble enzyme located in cytosol and releasedduring cell death, showed no obvious difference among ALS-, wt-, andcorrected-MNs or non-MNs at day-10 (FIG. 4A). Immunostaining for cleavedcaspase-3 showed no significant difference among the groups (FIG. 4B).These results suggest that the aggregate-containing MNs are surviving atthis stage.

In ALS patients and animals, axons degenerate before symptom onset andMN death (Bruijn et al., Science 281:1851-1854 (1998); Tu et al., Proc.Natl. Acad. Sci. U.S.A. 93:3155-3160 (1996); Fischer et al., Exp.Neurol. 185:232-240 (2004)). Indeed, we observed bead-like swellingsalong neurites of ALS MNs under phase contrast microscopy as early asday 7 after plating. At day 10, 25±2.4% neurites in ALS MN culturesexhibited bead-like structures, whereas only 5±0.4% of the neurites hadsuch structures in control MNs (FIGS. 4C, 4E). Few non-MNs exhibitedbeading structures (FIG. 4C). Aggregated NF as well as axonal NF isoften heavily phosphorylated (Goldstein et al., Proc. Natl. Acad. Sci.U.S.A. 80:3101-3105 (1983); Lee et al., J. Neurosci. 6:850-858 (1986)).Plasma phosphorylated neurafilament H (p-NF-H) levels closely reflectdisease progression and therapeutic response in the SOD1 (G93A) mice andare regarded as an ALS biomarker (Calvo et al., PLoS. One. 7:e32632(2012)). Indeed, immunostaining for p-NF-H showed dense staining in thebead structures in neurites of the ALS MNs, giving a dotted stainingappearance as opposed to an even staining pattern in control MNs ornon-MNs (FIG. 4D). Similarly, CHAT staining was concentrated in the beadformations (FIG. 4D) as it was weak in other areas of the ALS MNneurites. ELISA measurement of p-NF-H in culture media indicated thatits concentration in ALS MNs was 3-6 fold higher than that in the wt andcorrected MNs (FIG. 4F). The p-NF-H level in non-MNs across all groupswas very low (FIG. 4F). With extended cultures, we observed increasednumbers of beads along the neurites and fragmentation of the neurites(not shown), suggesting neurite degeneration at a later stage. Theseresults suggest that axons of ALS MNs undergo degeneration while thecell body is still structurally intact.

To further establish the cause/effect relationship between mutant SOD1and the above-described motor neuron pathology, we expressed the D90Amutant SOD1 or EGFP (control) in hESCs (H9 line) in the PPP1R12C (AAVS1)locus by TALEN-mediated homologous recombination. Western blottingrevealed a 2.8 fold increase in SOD1 protein in the SOD1-expressinggroup as compared to the EGFP control group after differentiation of thepluripotent cells to MNs and non-MNs (FIG. 5A). Expression of D90A SOD1or EGFP did not alter the differentiation of the hESCs to MNs ornon-MNs. Similar to the ALS cells, we observed progressively increasednumbers of NF aggregates in both cell bodies and neurites of MNs thatwere derived from the D90A SOD1-expressing, but not EGFP-expressing ESCsover time (FIGS. 5B-5D). However, no significant difference in NFaggregates was discerned in non-MNs between the D90A SOD1- and theEGFP-expressing groups (FIGS. 5C-5D).

Analysis of neurite degeneration indicated that 90±2% of neuritespresented bead-like structures in the D90A SOD1-expressing MNs whereasonly 4±0.3% of the MN neurites in the EGFP-expressing group had suchformations at day-10 (FIG. 5E). Few non-MNs exhibited beading structures(FIG. 5E). The p-NF-H in culture media was 6 fold higher inD90A-expressing MNs than in the EGFP-expressing MNs. By contrast, p-NF-Hwas barely detectable in non-MNs (FIG. 5F). Measurement of LDH in theculture media showed no significant difference among all the groups(FIG. 5G). These results indicate that expression of minimal amount ofmutant SOD1 (D90A) is sufficient to cause the same disease phenotypesthat are seen in cells with naturally occurring mutations.

Example 4 ALS Motor Neurons Exhibit Altered NF Subunit Proportion

Results of the neurite degeneration assays suggest that NF aggregationis an early and key event leading to MN axonal degeneration. NFs areassembled by copolymerization of NF-L, NF-M, and NF-H in a tightlycoordinated level (Hoffman and Lasek, J. Cell Biol. 66:351-366 (1975)).Transgenic disruption of their balance results in NF aggregation and MNdegeneration (Cote et al., Cell 73:35-46 (1993); Xu et al., Cell73:23-33 (1993)), resembling ALS pathology. By RT-qPCR analysis, wefound no difference in the expression of NF-H and NF-M mRNA between ALSand control (wt and D90D) MNs (FIG. 6A). Interestingly, NF-L mRNA wasreduced by 40-60% in ALS MNs as compared to control MNs (FIG. 6A). Innon-MNs, however, no obvious difference in all the three NF subunitmRNAs was observed between the disease and control groups (FIG. 6A).

At the protein level, Western blotting revealed that NF-H, NF-M, andNF-L were all decreased in ALS MNs but not in non-MNs as compared tocontrol cells (FIG. 6B). Moreover, NF-L was most prominentlydownregulated, representing only 30% of the level in wt MNs (FIG. 6B).Because NF-L was downregulated more than the other two subunits, theproportion of NF subunits was altered in ALS MNs but not in non-MNs(FIG. 6C).

Similarly, in the MNs derived from hESCs that express the D90A SOD1,mRNA levels of NF-L, but not NF-H and NF-M, were significantly decreasedas compared to controls (expressing EGFP). Strikingly, Western blottingrevealed downregulation of NF subunits, especially NF-L, thus alteringthe proportion of NF subunits in D90A SOD1- but not EGFP-expressing MNs.In contrast, expression of mutant SOD1 or EGFP had no effect on theexpression of NF subunits in non-MNs. These results further support theconclusion that mutant SOD1 alters NF compositions.

The above-described results suggest that mutant SOD1 alters theproportion of NF subunits, leading to NF aggregation and neuritedegeneration. To test this hypothesis, we conditionally expressed NF-Lor EGFP (control) in the PPP1R12C locus of D90A iPSCs by TALEN-mediatedhomologous recombination. As indicated by dose-dependent changes in EGFPintensity, the expression level of NF-L was increased in adose-dependent manner when doxycycline (DOX) was added to the MNcultures at day-21, as shown by Western blotting. Remarkably, inductionof exogenous NF-L, but not EGFP in the ALS MNs, not only increased theexpression of NF-L, but also NF-H and NF-M, to the level comparable tothat in corrected MNs (FIG. 7A). At 1 μg/ml DOX, the ratio of NF-L wasclose to that in wt or genetically corrected MNs (FIG. 7B). Importantly,the NF-L-expressing ALS MNs exhibited fewer NF aggregates in bothcytoplasm and neurites at day-10 after plating when compared to theEGFP-expressing ALS MNs, with approximately 30% and 50% reductions incytoplasm and neurites, respectively (FIGS. 7C-7D). Similarly, theproportion of bead-containing MN neurites was 12% in the NF-L-expressinggroup as compared to 25% in the GFP-expressing group even though it wasstill higher than that in the genetically corrected group (FIGS. 7E,7G). The p-NF-H in culture media in the NF-L-expressing ALS MNs wassignificantly lower than in the GFP-expressing ALS MNs (FIG. 7H).Together, these results indicate that induction of NF-L in ALS MNslargely restores NF subunit proportion, reduces NF aggregation, andmitigates neurite degeneration.

NF accumulation has been observed in ALS patients and transgenic animals(Carpenter, Neurology 18:841-851 (1968); Hirano et al., J. Neuropathol.Exp. Neurol. 43:471-480 (1984); Bruijn et al., Science 281:1851-1854(1998); Tu et al., Proc. Natl. Acad. Sci. U.S.A. 93:3155-3160 (1996),and transgenic alteration of NFs in neurons can cause ALS-like pathology(Cote et al., Cell 73:35-46 (1993); Xu et al., Cell 73:23-33 (1993)).Such similarity has led to a hypothesis that altered stoichiometry ofneuronal intermediate filaments results in ALS pathology (Julien andKriz, Biochim. Biophys. Acta 1762:1013-1024 (2006)). Usinggain-of-function (e.g., expressing mutant SOD1 in hESCs) andloss-of-function (e.g., genetic correction of D90A SOD1 mutation)analyses, we have now established unequivocally that mutant SOD1 leadsto NF aggregation. The sequential appearance of NF aggregation andneurite degeneration and especially the mitigation of neuritedegeneration following prevention of NF aggregation strongly suggestthat NF disorganization triggers the cascade of events, leading toaxonal degeneration in ALS MNs.

The key question is how NFs are disorganized in ALS MNs, leading toaggregation. It is known that over-expression of NF-H or NF-L leads toNF aggregation (Cote et al., Cell 73:35-46 (1993); Xu et al., Cell73:23-33 (1993)), highlighting the importance of correct proportion ofNF components for their physiological polymerization. In ALS patients,NF subunit proportion may be altered as there was one report showing a60% reduction in NF-L mRNAs in MNs using in situ hybridization (Bergeronet al., J. Neuropathol. Exp. Neurol. 53:221-230 (1994)) although noinformation is available whether the protein levels of NF subunits arealtered. Strikingly, we observed a significant and specific reduction ofNF-L but not NF-H or NF-M mRNA in mutant MNs. At the protein level, allNF subunits are downregulated but the NF-L is most significantly reducedto less than one-third of the level in wild-type MNs. Thus, it is thedown regulation of NFs and perhaps more importantly the alteredproportion of NF subunits that result in NF aggregation. This may appearcounterintuitive as MNs contain substantially higher amount of NFs thannon-MNs (see FIG. 6) and it is NF over-expression that results in NFaggregation and axonal degeneration in transgenic animals (Cote et al.,Cell 73:35-46 (1993); Xu et al., Cell 73:23-33 (1993)). Our presentstudy demonstrates that when the proportion of NF subunits is restoredby conditionally upregulating NF-L, NF aggregation and even neuritedegeneration are significantly mitigated in ALS MNs. We, therefore,propose that alteration of NF subunit proportion in ALS MNs leads to NFaggregation, which is a critical early step toward axonal degeneration.

Example 5 NF Aggregation and NF-L mRNA Down-Regulation in Sporadic ALSMNs

Skin fibroblasts were collected from 3 sporadic ALS patients who do nothave familial history of ALS. Induced PSC lines were obtained from theALS patients' fibroblasts using non-integrating Sendai virus.Differentiation of the ALS lines indicated that MNs and non-MNs can begenerated efficiently. Immunohistochemical staining for NF-H, NF-M, andNF-L showed NF aggregations in MNs but not in non-MNs (FIG. 8),highlighting the recapitulation of disease hallmarks by the iPSC model.RT-qPCR analysis indicated that NF-L mRNA, but not NF-H mRNA or NF-MmRNA, was down-regulated in MNs but not in non-MNs that weredifferentiated from sporadic ALS iPSCs for 31 days (day-10 after platingday-21 cells) (FIG. 8, MN data are shown). Such a striking similarity tothe phenotypes observed in SOD1 familial ALS MNs highlights a commonmechanism underlying MN degeneration.

Example 6 NF Aggregates Observed in MNs from Sporadic ALS Subjects andALS Subjects Having a TDP43 Mutation

To determine if NF aggregation occurs in ALS patients with othermutations or those without known genetic defects, we established iPSCsfrom a patient with TDP43 (TAR DNA-binding protein 43) mutation (G298S)and two sporadic ALS patients (no known genetic defects) Sendai virus.As seen with SOD1 mutant ALS cells, iPSCs from TDP43 or sporadic ALSpatients differentiated to motor neurons with similar efficiency.Interestingly, immunostaining for NF-H, NF-M and NF-L indicated that theboth TDP43 and sporadic ALS MNs contained distinct, inclusion-like focalaccumulation of immunoreactive products (FIG. 12). The aggregates werepresent both in cell body and neurites. The number of NF inclusions inboth cell body and on neurites increased over time. The rates ofneurofilament aggregation were significantly higher in ALS MNs comparedto that in non-ALS MNs (P<0.05) (FIG. 12). The average size of each NFinclusion was approximately 2.94−3.72×10⁻⁴ mm² in cell bodies andapproximately 3.75−5.53×10⁻⁶ mm² in neurites at Day 10.

Together, these data demonstrate that NF aggregation is a common motorneuron pathology to all ALS patients (those having genetic defects andthose without known genetic defects). These data also suggest there is acommon underlying mechanism for ALS pathogenesis.

We claim:
 1. A method of diagnosing neurodegenerative disease in asubject, comprising the steps of (a) obtaining induced pluripotent stem(iPS) cells from somatic cells of a subject, wherein the iPS cells arecapable of differentiation into neurons; (b) culturing the iPS cellsunder conditions suitable to differentiate the iPS cells into neurons;and (c) detecting an indicator of neurofilament aggregation or neuritedegeneration in the iPS cell-derived neurons, wherein increasedneurofilament aggregation or neurite degeneration relative to neuronsderived from iPS cells of an individual not having neurodegenerativedisease indicates that the subject has a neurodegenerative diseaseselected from the group consisting of amyotrophic lateral sclerosis(ALS), Spinal Muscle Atrophy (SMA), and Charcot-Marie-Tooth disease(CMT), and thereby diagnosing neurodegenerative disease in the subject.2. The method of claim 1, wherein the iPS cell-derived neurons are motorneurons.
 3. The method of claim 1, wherein detecting neurofilamentaggregation comprises determining a level of NF-L polypeptide or NF-LmRNA in the subject's iPS cell-derived neurons.
 4. The method of claim1, further comprising detecting a level of phosphorylated neurofilamentin a biological sample of the subject.
 5. The method of claim 4, whereinthe biological sample of the subject is cerebrospinal fluid.
 6. A methodof detecting a neurodegenerative disease in a subject, comprising thesteps of: (a) measuring a level of NF-L in neurons derived from iPScells obtained from somatic cells of a subject; (b) relating the levelto a reference level of NF-L; and (c) thereby detectingneurodegenerative disease in the subject based on a reduced level ofNF-L relative to the reference level.
 7. The method of claim 6, whereinthe neurodegenerative disease is selected from the group consisting ofamyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease (CMT),and Spinal Muscle Atrophy (SMA).
 8. The method of claim 7, wherein theneurodegenerative disease is ALS, wherein the neurons are motor neurons,and wherein the reference level is a level of NF-L mRNA in motor neuronsderived from iPS cells of an individual having ALS.
 9. The method ofclaim 8, wherein the reduced level of NF-L mRNA is at least 50% lowerthan the reference.
 10. A method for treating a neurodegenerativedisease in a subject in need thereof, comprising administering one ormore recombinant polynucleotides encoding NF-L to the subject, whereinthe polynucleotides are targeted to neurons, and wherein expression ofNF-L in targeted neurons treats the neurodegenerative disease.
 11. Themethod of claim 10, wherein the neurodegenerative disease is selectedfrom the group consisting of amyotrophic lateral sclerosis (ALS),Charcot-Marie-Tooth disease (CMT), and Spinal Muscle Atrophy (SMA). 12.The method of claim 10, wherein the polynucleotides are administered ina vector.